FI63596C - MICROBIA DIAGNOSIS FOERFARANDE SOM GRUNDAR SIG PAO SKIKTSHYBRIDISERING AV NUCLEINSYROR OCH VID FOERFARANDET ANVAENDA KOMBINATIONER AV REAGENSER - Google Patents

Abstract

Sandwich hybridization of nucleic acids can be used for the identification of microorganisms or groups of microorganisms present in a sample. The technique can be used to identify, from a single sample containing nucleic acids of the microorganisms or groups of microorganisms to be identified, after first rendering the said nucleic acids single-stranded, all the microorganisms or groups of microorganisms by adding to the sample a pair of nucleic acid reagents for each microorganism or group of microorganisms to be identified, of which pair one nucleic acid reagent is attached to a solid carrier in the single-stranded form and the other contains a completely different nucleic acid fragment from the same microorganism or group of microorganisms, which fragment is labelled with a marker. The nucleic acid fragments of a pair hybridize to a complementary single-stranded nucleic acid from the sample and the hybrid thus formed on the solid carrier is labelled with the marker. Because the labelled nucleic acid reagent does not hybridize directly with the reagent attached to the solid carrier, only those carriers having attached nucleic acid reagents corresponding to nucleic acids in the sample become labelled and the presence of such carrier-bound labels can be measured using established methods.

Description

63596

Microbial diagnostic method based on layer hybridization of nucleic acids and combinations of reagents used in the method 5

The invention relates to a microbial diagnostic method based on layered hybridization of nucleic acids on a solid support for the simultaneous detection of one or more microbes and / or microbial groups in a single undivided sample and to a series of reagent combinations used in the method.

15 In traditional microbial diagnostics, the presence of a microbe in test samples has been demonstrated by isolating the microbe in question. After enrichment culture, the microbe is identified either by its biochemical properties or by immunological methods. Such diagnostics 20 require that the microbe in the sample be reproducible. In addition, detection through isolation is a laborious method which, in the case of viruses, may take 4 to 6 weeks.

It is an object of the invention to provide a diagnostic method in which the presence of a microbe in a sample is detected by identifying its genome, nucleic acid, by sensitive and specific nucleic acid hybridization. Nucleic acid hybridization per se is an old and known method for studying the identity of nucleic acids. Opposite-branded nucleic acid strands have the ability to form a tight two-stranded structure based on the mating rule of the counterparts, and the resulting hybrid can be distinguished from the single-stranded nucleic acid.

2 63596

Methods based on nucleic acid identification have so far been applied to microbial diagnostics to some extent. Enterotoxigenic E. coli has been detected in faecal samples by colony hybridization 5 using the gene responsible for toxin production as a probe.

Positive hybridization is detected by autoradiography (Moseley, S.L. et al. J. Infect. Dis. (1980) 142, 892-898). Colony hybridization is based on the method originally developed by Grunstein and Hogness (Proc. Natl. Acad. Sci. USA (1975) 72, 3961-3965). In addition, hybridization has been applied to distinguish between Herpes simplex 1 and Herpes simplex 2 viruses (Brautigam A.R. et al. J. Clin. Microbiol. (1980) 12, 15226-234), but not as a rapid diagnostic method but after concentrate culture in virus typing. In this method, a soluble double-stranded hybrid is separated from single-stranded hybrid by affinity chromatography.

20

Recently, work has been done in which DNA from cells infected with Epstein-Barr virus, i.e., a sample, is directly attached to filters after certain treatments. The nucleic acid in question is identified by hybridizing the filters in the presence of a radioactive probe and positive hybridization is detected by autoradiography (Brandsma, I. & Miller, K. (1980) Proc. Natl. Acad. Sci. USA 77, 6851-6855).

30 In the Lancet 10 Det., P. 765-7, 1982 discloses a similar method in which a radiolabeled probe detects hepatitis B virus from a sample whose DNA has been made single-stranded and attached to a nitrocellulose filter. DE 2950295 describes a method for the preparation of such a hepatitis B virus radiolabeled probe using recombinant DNA technology.

3 63596

The method of our invention uses a series of combinations of reagents consisting of at least two different nucleic acid reagents prepared from different parts of the genome of the same microbe or group of microbes.

5 Of these nucleic acid reagents, one is bound to a solid support and the other is labeled with a label.

These two nucleic acid reagents can be used to directly detect all microbes and groups of microbes for which reagents have been prepared from a single-stranded DNA sample. The use of a kit of two reagents according to our invention makes the diagnostic method considerably more specific than the method described above and also allows the construction and simultaneous use of different kit combinations without interfering with each other and the specificity of the test.

The present invention is based on the layer hybridization technique (Dunn, A.R. & Hassell, 3.A. (1977) Cell 12, 20 23-36), which simplifies sample handling and hybrid detection. For this reason, the technology is excellently suited for diagnostic use.

In the method of the invention, all desired microbes or microbial groups can be identified without dividing from one and the same sample containing nucleic acids of single-stranded microbes or microbial groups to be diagnosed by adding two nucleic acid reagents for each microbial or microbial group to be identified. These nucleic acid reagents contain completely different but preferably closely spaced nucleic acid fragments from the same microbe or 63596 microbial clusters, which can be prepared either directly from the microbial genome or by recombinant QNA-5 techniques known per se, and one of these nucleic acid fragments is isolated. and attached to a solid support, preferably a nitrocellulose filter, and the other is monofilamented and labeled with a label suitable for the method. When these nucleic acid reagents, two different for each microbial or group of microbes to be identified, are contacted with the single-stranded nucleic acids to be identified in the sample, these nucleic acids bind to the corresponding nucleic acid fragments on the solid support. fragments. These labeled nucleic acid fragments alone do not hybridize to solid support nucleic acid fragments but to single-stranded nucleic acids from a sample. In this case, only those solid supports to which the corresponding nucleic acids in the sample are bound are labeled, and these solid supports can be rinsed and the labeling measured by methods known per se.

The method according to the invention can in principle be used for the identification of all organisms containing nucleic acids, either DNA or RNA, such as, for example, viruses, bacteria, molds and yeasts. A particular advantage of this method is that the same sample can be tested simultaneously for the presence of all relevant bacteria and viruses, regardless of whether the microbes contain DNA or RNA. That is, by combining the reagents 35, "kit" assemblies can be constructed so that each identifiable microbe has its own solid support and labeled nucleic acid reagent with 5,63596. All filters in the reagent combination can be added to the sample at once, as well as labeled nucleic acid reagents. Once hybridization has taken place, the solid supports are washed and labeling is measured, and only the correct solid support is labeled, i.e. the one with the corresponding microbial genome in the sample.

10 The method and a series of combinations of reagents can be used e.g. in medical microbiology, veterinary microbiology, food hygiene research and microbial diagnostics of plant diseases. All homogenates from animal or plant tissue, patient secretions such as blood, faeces, nasopharynx and urethral mucus are suitable as samples. It can be estimated that the method is sensitive enough to detect microbial counts normally present in clinical specimens. Pre-enrichment of the microbe in the sample by culture is, of course, possible before performing the identification test and in some cases it is absolutely necessary. The method is also suitable for examining samples from which the microbe is no longer cultured but the microbial constituents are still abundant in the sample (eg antibiotic treatment has been started) or microbial culture is a particularly laborious and difficult procedure (eg anaerobic bacteria abundant in wet samples). in the case of anaerobic infection).

30

The method can be applied in microbial diagnostics of clinical medicine and food hygiene, e.g. to the identification of the following microbes or groups of microbes.

6

Respiratory infections: 63596 a) Bacteria: ^ -hemolytic streptococci, (group A), Haemophilus influenzae. Pneumococci, Mycoplasma 5 pneumoniae, mycobacteria b) Viruses: Influenza A, Influenza B, Parainfluenza 1-3, Respiratory syncytial virus, adenoviruses, coronaviruses, rhinoviruses 10

Diarrhea: a) Bacteria: Salmonella, Shigella, Yersinia entero-colitica, enterotoxigenic E. coli, Clostridium 15 di fficile, campylobacter b) Viruses: rotaviruses, parvoviruses, adenoviruses, enteroviruses 20 Sexually transmitted diseases: a) Bacteria: Neisseria gonorrhoea Chlamydia trachomatis 25 b) Viruses: Herpes simplex virus c) Yeasts: Candida albicans d) Protozoa: Trichomonas vaginalis 30

Sepsis: a) Bacteria: γ-hemolytic streptococci, (group A), Pneumococci, enterobacteria as group 7 63596

Food hygiene: (a) Bacteria: Salmonella and Clostridium perfringens 5 Depending on the choice of reagents, the specificity of the test may be limited to microbes (eg salmonella) or by selecting recognizing reagents for an entire microbial group from a common gene region.

10

The nucleic acid reagents required in the layer hybridization technique of the invention are produced by recombinant DNA technology. The reagents produced for Example 1 and the performance of the test are described below.

15

reagents

Adenovirus 2 (strain stored in KTL, i.e. the Public Health Institute in Helsinki) is grown and purified and DNA is isolated (Petterson, U. & Sambrook, J. (1973) J. Mol. Biol. 73, 125-130) (referred to as Ad2 DNA). .

The DNA is digested with the restriction enzyme BamHI (BRL, or Bethesda Research Laboratories), which cleaves the DNA into four identical fragments. Of these four, two fragments are ligated to the BamHI cleavage site of the vector selected as the vector pBR322 (BRL) using the T4 ligase enzyme (BRL). (The fragments were not separated before ligation, but the additional insert received by the plasmid - i.e. the insert - is not identified until after cloning.) Then plasmid DNA, which now includes so-called recombinant plasmids, i.e. molecules that have accepted adenovirus DNA sequences as part of it. as desired, is transferred to a bacterial host (E. coli HB101 (K12) (ga1, pro, Leu, hrs, hrm ~, recA, 35 str, F) (from KTL) by transformation (Cohen, SN et al. (1972 ) Proc. Natl. Acad. Soi. USA 69, 2110-2114) Among the transformed bacterial clones, those most likely to contain the recombinant plasmid are selected, the bacterium obtains from plasmid pBR322 and __ - 1, .. .... - β 63596 ampicillin and tetracycline resistance genes (Bolivar, F. et al. (1977) Gene®, 95-113) Bacteria containing the recombinant plasmid, on the other hand, are sensitive to tetracycline because the BamHI enzyme cleaves the tetracycline resistance of plasmid DNA. from the region and the foreign DNA associated with that region destroys the gene. The insert obtained by the plasmid is characterized after plasmid enrichment by BamHI digestion by determining the size of the insert by agarose gel electrophoresis. Adjacent BamHI D and C fragments of 10 Ad2 DNA (cf. gene map) are selected as reagents (Söderlund, H. et al. (1976) Cell 7, 585-593). The correct recombinant plasmids, Ad2C-pBR322, KTL No. EH231 and Ad2D-pBR322, KTL No. EH230, are grown and purified as described (Clewell, DB and Helinski, DR (1969) 15 Proc. Natl. Acad. Sci. USA). 62, 1159 - 1166).

The recombinant plasmid Ad2D-pBR322 is used as such as a filter reagent. Plasmid sequences do not need to be deleted for this purpose because there are no 20 pBR322 sequences in the sample. Instead, the radiolabeled nucleic acid reagent Ad2_BamHI C fragment is separated from pBR322 DNA after BamHI digestion by agarose gel electrophoresis. The C fragment is isolated from an LGT agarase (Marine Colloids, Inc.) gel by phenol-25 extraction or electroelution (Wieslander, L.

(1979) Anal. Biochem. 9_8, 305-309) and concentrated by precipitation with ethanol.

It is highly convenient to further clone the nucleic acid fragment to be selected as the labeled nucleic acid reagent into another vector to eliminate hybridization from the background due to direct hybridization to the filter of plasmid sequences remaining contaminated in the labeled nucleic acid reagent. One such second optical vector is the single-stranded DNA phage M13 mp7 (BRL), to which DNA fragments obtained, for example, by the BamHI enzyme are easily transferred (Messing, 0. et al.

(1981) Nucleic Acids Res. £, 309 - 323).

9 Attachment of DNA to filter 63596

The recombinant plasmid Ad2Ö-pBR322 is denatured into a single strand and cleaved blindly at a few sites by 0.2 N 5 NaOH treatment (5 min at 100 ° C), after which the DNA is cooled and neutralized and pipetted into the transfer solution 4 x SSC medium on ice (SSC = 0.15) just before transfer to the filter. M NaCl, 0.015 Π Na citrate). Filters (Schleicher & Schull ΒΑΘ5 ni cellulose) are carefully wetted in 4 x SSC solution (approximately 2 h) before DNA application. The DNA is adhered to the filter in a dilute solution (0.5 to 1 ug / ml) by filtering the solution through the filter under gentle water suction, whereby the DNA adheres. The filter is capable of binding 0NA at about 1Θ0 μg / cm (Kafatos, F.C.

15 et al. (1979] Nucleic Acids Research 7, 1541-1552). DNA concentrations of 0.5 μg DNA / 2.5 cm diameter filter up to 1 μg DNA / 7 mm diameter filter are used. After DNA filtration, the filters are washed 4x with SSC, dried at 20 room temperature and last treated in a +80 ° C vacuum oven for 2 h, after which the DNA remains stable and the filters can be stored at room temperature for long periods (Southern, EM (1975)). 0. Mol Biol 96, 503 - 51 7).

25

Labeling of a radioactive nucleic acid fragment 125

The I isotope is used as the radiolabel.

30 This is detectable with the Y ~ counter that exists in most large laboratory units. The half-life of the isotope is 60 days, which is why the shelf life of the reagents labeled with this is about 4 months.

1 G

63596 “Nie k-translation” stamp

The idea of the method is to change one of the nucleotides of the nucleic acid to radioactive, at the same time labeling all the DNA. 5 This occurs in Rigby, P.W.3. et al. (1977) 3. Mol. Biol. 113, 237-251). In the reaction, the DNA is radiolabeled when the solution contains 125 I-isotope-labeled dedxinucleoside triphosphate as raw material, in this case I-dCTP (Radiochemical Center 10, Amersham:> 1500 Ci / mmol). Under optimal conditions, up to 10 cpm of DNA per DNA can be labeled. Labeled DNA is purified from nucleotides released in the reaction by simple gel filtration, e.g., using BioGel P30 (BioRad).

15

Other stamping

The single-stranded nucleic acid reagent produced in M13 mp7 phage is conveniently labeled by direct iodination, with reactive I being hard-ligated to the nucleic acid (Commerford, SL (1971) Biochemistry _1_0, 1993-2000, Orosz, JM and Wetmur, 3). G.

(1974) Biochemistry j_3, 5467-5473). Alternatively, the nucleic acid can be radioactivated by attaching a radioactive nucleotide to its ends by terminal transferase (Roychoudhury, R. and Wu, R. (19B0) Meth. Enzymol. 6_5, 43-62).

30 The reagent preparation discussed above concerns microbes whose genetic material is DNA. Cloning of RNA virus genomic fragments is accomplished by first making a DNA copy (cDNA) of the viral RNA using a reverse transcriptase enzyme, after which the DNA polymerase renders the DNA double-stranded and the DNA can be cloned as described above (Salser , W. (1979) in Genetic Engineering, Ed. AM Chakrabarty, CRC Press, pp. 53-81).

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For each microbe, the most appropriate cloning method is used, in which the vector used and the host may vary. Possible λ-phage vector, other plasmids, cosmids, cloning 5 mm. In Bacillus subtilis, etc. (Recombinant DNA, Benchmarck Papers in Microbiology, Vol. 15, Ed. K.3. Denniston & LW Enqvist, Dowden, Hutchinson & Ross, Inc. (19BDj Ish-Horowicz, D. and Burke, 3 .F (19813 Nucleic Acids Research j), 2989 - 29983.

10

Test performance Sample handling 15

The microbinucleic acid to be examined must be released both inside the microbial and from the infected cells and denatured into a single-stranded form. Viral genomes are released by treating the sample material with 1% sodium dodecyl sulfate solution (SDS) and degrading their protective proteins by proteinase K treatment (1 mg / ml + 37 °, 60 min). In addition, the bacterial sample must be digested with lysozyme and EDTA.

25 The 3s sample contains a lot of tough giant cellular DNA, this must be cut from here to here to reduce the viscosity, e.g. by sonicating or squeezing the sample through a thin needle a few times.

30

hybridization

Hybridization occurs, for example, in 50% formamide (deionized, stored at -20 ° C), 4 x SSC, 1 x 35 Denhardt's solution (Denhardt, D.T. (1966) Biochem.

Biophys. Res. Commun. 2, 641-646) with 1% SDS weather and 0.5 mg / ml DNA (salmon sperm or calf thymus) at + 37 ° C 12,63596, usually overnight for 16-20 hours. The filters selected for the test are incubated in a suitable vessel into which the hybrid * solution is pipetted and hybridization is started. The hybridization * solution consists of (a) a pretreated sample and associated radioactive nucleic acid reagent (s) denatured by boiling for 5 minutes, after which the solution is rapidly cooled to 0 ° C; (b) concentrated formamide, SSC and Denhardt * solutions pipetted into the denatured and cooled mixture (a). After mixing, the hybridization solution is pipetted into the hybridization vessel for filters. After hybridization, the filters are washed thoroughly and the filters are counted one by one in a V-counter.

The invention is further clarified by a few examples of use.

Example 1

Detection of adenovirus by layer hybridization method 20 (Table 1)

Details of the test can be found in the table text. The layer hybridization method is able to detect viral DNA from solution, but it is equally possible to detect the viral genome from infected cells.

The background of the hybridization is measured in a tube to which no sample has been added but only the filter and the labeled nucleic acid reagent. The background is due to the pBR322 sequences contained in the labeled nucleic acid acid reagent, which hybridize directly to the filter without a mediating sample. The calf thymus and empty filters used as controls in the test describe, on the one hand, the specificity of the hybridization and, on the other hand, the magnitude of the non-specific background due to, for example, insufficient washing.

In the following tables, the background due to reagents has been subtracted from the cpm values hybridized to the filters.

Table 1 13 63596

Adenovirus Test 5 Sample Filters (cpm)

Adeno 1) Calf thymus 2) Blanko 3)

Adenovirus 2-DNA (BRL) 10 (500 ng) 9000 49

Adenovirus-infected HeLa cells (6 x 10 ^) 8200 15

The filters; 13 Ad2D-pBR322 plasmid 2 yug 2) Calf thymus DNA 1 yug (Boehringer Mannheim) 3) Blanko Cei DNA) 20

Labeled nucleic acid reagent;

Ad2_BamHI C fragment purified, specific activity 90 x 10 ^ cpm / μg (200000 cpm ^ "'i / reaction) Hybridization:

50% formamide, 4 x SSC

Denhardt's solution with 0.5 mg / ml salmon sperm DNA and 1% SDS, + 37 ° C, 16 h 30 Wash; 0.1 x SSC, at room temperature 40 min Samples;

Adenovirus 2-DNA (BRL) 35 Adenovirus, type 2 infection occurs in HeLa cells. The cells are lysed by 1% SDS treatment, followed by the addition of proteinase K enzyme (Sigma) 1 mg / ml and allowed to act at + 37 ° C for 30 min. Prior to denaturation, the sample is squeezed through a thin needle.

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From the values in the table, the background caused by the reagents obtained from the corresponding hybridization without sample has been subtracted.

5 Example 2 Detection of RNA virus by the layer hybridization method (Table 2) 10 The RNA virus model used is Semliki Forest virus (prototype strain obtained from the London School of Hygiene and Tropical Medicine) whose genome is single-stranded RNA.

Using the viral genome as a template, cDNA is prepared and cloned into plasmid pBR322 at its PstI cleavage site as described by Garoff et al. have described (Proc. Natl. Acad. Sci. (1980) USA 77 6376-63880). The resulting recombinant plasmid is pKTH312 KTL No. EH 232. Its viral genome-derived portion is about 1400 nucleotides in length and originates in the region of viral structural proteins, from about nucleotide 200 to nucleotide 1600 when numbering begins at the beginning of structural genes (Garoff, H. et al. 1980 ). For reagent preparation, the entire recombinant plasmid pKTH312 is linearized with EcoRI restriction enzyme (BRL) (sequences derived from Semliki Forest virus do not have 25 EcoRI recognition sites), which in turn is cleaved into two fragments by the XhoI site (BRL). The larger EcoRI-XhoI fragment A (about 3900 bp in length) is attached to the filter when the smaller fragment 125 B (about 1850 bp in length) is labeled with I by nick translation.

Both whole Semliki Forest virus and Semliki Forest virus-infected cells are used as samples in the experiment. 35 In both cases, the virus-specific nucleic acids in the sample consist exclusively of RNA.

Table 2 15 63596

Detection of Semliki Forest virus by layer hybridization 5__ Sample Filters (cpm)

Semliki Forest Calf thymus 2) Blanko 3) virus 1) 10 Semliki Forest virus 30 ^ to 3340 - 33

Semliki Forest virus infected cells (5 x 105) 2698 8 10 15

Uninfected cells 10 5 8

The filters; 1) EcoRI-XhoI fragment A of plasmid pKTH312 A 1.2 [mu] g 2) Calf thymus DNA 1 / [mu] g 3) Blank (no DNA)

Labeled nucleic acid reagents: EcoRI-XhoI fragment B of plasmid pKTH312, specific c λ y c activity 90 x 10 cpm // ug DNA (200,000 cpm I / reaction)

hybridization:

As in Example 1 30

Washing;

As in Example 1 Samples; 35 Semliki Forest virus 30 μg is digested. With SDS before the test. Infected cells are treated as described in Example 1. Semliki Forest virus infection is performed in BHK-21 cells.

i 'l 16 63596

From the values in Table 2, the reagent-induced background obtained from the corresponding hybridization without sample has been subtracted.

5 Example 3

Virus sample in which viral messenger RNA is detected by the layer hybridization method (Table 3) 10 Layer hybridization reagents are prepared from SV40 virus DIMA (BRL) by cutting the DNA into two pieces.

Using the PstI enzyme (Baehringer Mannheim) (Lebowitz, P.

and Weissman, S.M. (1979) Curr. Topics in Microbiol.

Immunol, 8-7, 43-172) and fragments are isolated and purified

15 by agarose gel electrophoresis. Fragment A

(4000 bp) is radiolabeled in nick-trans-125 with I and fragment B (1220 bp) is attached to a filter.

The ONA fragments are selected so that both have regions encoding both late and early transmissions. Thus, B contains about 700 bases of the structural protein VP1 gene and more than 600 bases of the early messenger gene. Because SV40 virus DNA itself is a covalently closed 25 ring, it is not suitable as a sample in the assay without being opened linearly. Thus, by using infected cells as a sample, it can be tested how well the method is suitable for detecting RNA copies made according to the viral genome. Table 3 shows that the test is suitable for examining 30 excellently infected cells. It also demonstrates that the same reagents can be used to examine both the viral DNA itself and the resulting messenger RNAs.

63596

Table 3 1 7 Detection of SV40 viruses by layer hybridization procedures · sample 5 Filters (cpm) SV40 1) Calf thymus 2) Blanko 3)

Experiment 1 SV4Q virus DNA (50 ng) 10 (linearized) 20061 159 104

No sample - 1 5 Experiment 2 CV1 cells infected with SV40 virus 40 h after infection (10 ^ cells) 30814 294 530 20 Uninfected cells -

Filters: 1) Shorter fragment of SV40 virus circular DNA cleaved by PstI restriction enzyme PstI B 0.2 yug 2) Calf thymus DNA 1 / Jg 3) Blanko (no DNA)

Labeled Nucleic Acid Reagent: Longer PstI A fragment from 30 sV40 viral DNA, specific activity 28 x 106 cpm // Jg DNA (200,000 cpm 125I / reaction)

hybridization:

As in Example 1, the hybridization time is 40 hours

Washing;

As in Example 1 18 63596 Samples: SV40 viral DNA (BRL) is linearized with EcoRI restriction enzyme (BRL). CV1 cells (Biomedical Center, Uppsala University) are infected with SV40 virus (obtained from Janice Y.

5 from Chou & Robert G. Martin, NIH, Bethesda) and cells are harvested 48 hours after infection. Sample processing is performed as in Example 1.

From the values in Table 3, the background caused by the reagents obtained from the corresponding hybridization without sample has been subtracted.

Example 4 Detection of Bacillus amyloliquefaciens by the layer hybridization method (Table 4)

As reagents, fragments of the o (-amylase gene) of B. amyloliquefacjens * in E18 (VTT or State Technical Research Institute) isolated from the recombinant plasmid pKTH10 for this test have been used (Palva, I. et al., (19Θ1) Gene, V5 43 .-5 T) after restriction enzyme treatment by agarose gel electrophoresis The fragments used for the assay are the 0 (-amylase gene region ClaI> EcoRI fragment, (460 25 bp) (Clal Boehringer Mannheim) and the EcoRI-BamHI fragment ( Of these, the EcoRI-BamHI fragment is attached to a filter and the ClaI-EcoRI fragment is labeled with 1 * L-li isotope 11a radioactively by nick translation.

30

Table 4 shows that by the layer hybridization technique, B. amyloliquefaciens can be identified in the sample by one (based on the X-amylase gene. E. coli was negative in the test (gave a result comparable to 35 in the background).

19 63596

Table 4

Bacterial diagnostics by layer hybridization 5 Sample Filters (cpm) 0 (-amylase 1] Calf thymus 2) Blanko 3)

pKTHlQ plasmid DNA

(linearized) 1 μg 5773 47 10

No sample - E. coli HB101 (109) - - 1 5 Bacillus amylolique- faciens (3 x 10) 3377

Bacillus amylolique- faciens (109) 2871 20 _I___

The filters; 1) Plasmid pKTH10 (EcoRI-BamHI fragment of the X-amylase gene 0.35 μg 25 2) Calf thymus DNA 1 μg 31 Blanko (no DNA)

Labeled nucleic acid reagent: ClaI-EcoRI fragment of the CV-Amy lasal gene of the pKTHIO plasmid, 6 125 30 specific activity 35 x 10 cprrv ug (200,000 cpm I / reaction)

hybridization:

As in Example 1 35 Washing:

As in Example 1 20 63596 Samples:

Bacterial samples are treated with lysocyr (1 μg / ml) for 30 min at + 37 ° C; 5 mM EDTA is also added to the E. coli sample. At the end of the treatment, SDS (2%) is added to all samples, the samples are pressed through a thin needle twice to reduce the viscosity and denatured by boiling as described in the text during sample processing.

From the values in Table 4, the background caused by the reagents 10 obtained from the corresponding hybridization without sample has been subtracted.

Example 5 Example of a combination of reagents acting as a "putty" based on the layer hybridization method (Table 5) As samples in this test, cells infected with three different viruses (adenovirus, SV40 virus and Herpes 20 simplex virus) and Bacillus amyloliquefaciens are all sampled into each sample. reagents: 1 filter containing SV40 virus, a filter containing 25 adenovirus, Bacillus amyloliquefaciens am-amylase gene and calf thymus DNA and one empty filter, in addition to 200000 cpm of each of the following labeled nucleic acid reagents: SV40 virus and adenovirus 0 (-amylaasigeeni DNA reagents,

Our example shows that an appropriate set of microbes can be tested on a sample at the same time without diluting and dividing it by mixing the reagents in the sample. Both viral and bacterial nucleic acid can serve as samples. The identification of the microbe in the sample is easy because it is done on the basis of filters marked 35.

Table 5 21 63596

Kit based on the layer hybridization method 5 Sample Filters (cpm) SV40 1) Adeno 2) 0 (-arry- Calf Blanko 5) glass 3) thymus 4) SV40 virus irifinfected cells MO6) 16390 2 13 22 31

Cells infected with Adeno-2 virus (6 x 105) - 6750 5 13 15

Cells infected with the herpes simplex virus MO6) - 5 13 20 Bacillus amylolique- faciens MQ9) 15 8 6500 16 5

Uninfected cells 25

The filters; 1) as in Example 3 2) as in Example 1 3) as in Example 4 30 4) Calf thymus DNA 1 yug 5) Blanko (no DNA)

Labeled nucleic acid reagents: SV40 virus as in Example 3 35 Adenovirus as in Example 1 (X-Amy glass gene as in Example 4 22

hybridization; 63596

As in Example 1 Washing: 5 As in Example 1 Samples: SV40 virus infected cell samples are described in Table 3. Adenovirus infected cell samples 10 are described in Table 1.

0

Herpes simplex, a type 1 virus, infects 10 Vero cells. Cells are harvested after 20 hours of culture, when microscopic signs of viral infection can be detected in the cells. The sample is treated in the same way as adenovirus-infected cells (Example 1).

Sample of Bacillus amyloliquefaciens:

As in Table 4 20

From the values in Table 5, the reagent-induced background obtained from the corresponding hybridization without sample has been subtracted.

Il 63596 23 5

Example 6

Detection of Escherichia coli by the layer hybridization method 11a (Table 6)

Test reagents were prepared from fragments of the E. coli ompA gene. OmpA (outer memhrane protein A) is an outer membrane protein of E. coli.

10 The plasmid pTU100 from the SITRA recombinant DNA group (Pauli Kallio, master's thesis, Department of General Microbiology, University of Helsinki) (Henning et al. (1979), Proc. Natl. Acad. Sci. USA 7_6, 4360-) was used as starting material. 4364) processed hybrid plasmids pKTH40 and pKTH45.

Plasmid pKTH45 (deposited with KTL No. EH254) containing 740 base pairs from the 5 'end of the ompA gene fused to plasmid pBR322 was used directly as a filter reagent.

Plasmid pKTH40 contains about 300 base pairs from the 3 'end of the ompA gene, immediately followed by about 1400 base pairs from the rest of the E. coli genome. pKTH40 was digested with the restriction enzyme BamHI to give a DNA fragment from E. coli of a total of 1700 25 base pairs. It was transferred to single-stranded bacteriophage M13mp7 (Messing et al. (19B1), Nucl. Acids Res. E, 309-321, Heidecker et al. Acids Res., 2871-286Θ). The resulting recombinant mage phage, mKTH12Q7 (deposited on the KTL with those EH256), was used in layer hybridization as a probe by labeling it with a J-isotope radioactive as described in "Other Labeling" on page 9.

As shown in the assay (Table 6), both DNA from disrupted E. coli cells and pure DNA isolated from E. coli can be detected by a layer hybridization method.

Table 6 24 63596

Detection of Escherichia coli by the layer hybridisation method · __ Sample Filters (cpm) ompA 1) Calf thymus 2) Pig 3) E. coli K12 HB101'DNA 10 a) 2 x 107 282

E. coli K12 HB101 DNA

a) 2 x 108 2206 15 E. coli K12 HB101 cells b) 2 x 107 1113 E. coli K.12 HB101 cells b) 2 x 10® 2327 12 5 20 _____ a) Number of DNA molecules, b) Number of cells

Filters: 25 1) pKTH45 plasmid 1.088 μg (2 x 10 ^ molecules) 2) Calf thymus DNA 1.088; ug 3) Blanko (no DNA)

Labeled nucleic acid reagents; 30 mKTH 1207, specific activity 8 x 107 cpm / μg DNA (200,000 cpm / reaction)

hybridization; 4 x SSC, 1 x Denhardt without BSA (bovin Serum album),

0.25% SDS, 200 ug / ml Herring sperm DNA, 17.5 h, ♦ 65 ° C

Washing;

As in Example 1 63596 Samples: E. coli K12 HB101 DNA was isolated by the Marmur method (Marmur (1961), J. Mol. Biol. 3, 208-21Θ). DNA denaturation for the assay was performed with 7 mM NaGH, 100 ° C, 5 min.

Cells were lysed by treatment with lysozyme (500 μg / ml), EDTA (70 mM, + 37 ° C, 30 min), SOS (0.25%, + 65 ° C, 15 min) and the released DNA was denatured with alkaline soup (14 mM NaOH, + 100 ° C, 5 min).

From the values in Table 6, the background caused by the reagents obtained from the corresponding hybridization without sample has been subtracted.

Claims (10)

26 63596 A solid-state microbial diagnostic method based on layered hybridization of nucleic acids to simultaneously identify one or more microbes and / or groups of microbes, comprising two different nucleic acid reagents, one comprising a single-stranded nucleic acid , characterized in that a series of at least two different nucleic acid reagents for each microbial and / or microbial group to be identified, one bound to a solid support and the other labeled with a label known per se, is contacted with one of the undivided samples. with single-stranded nucleic acids of microbes or groups of microbes in a manner known per se, in which case the nucleic acid from sample 20 the nucleic acids from which the corresponding nucleic acid reagents are used hybridize to the corresponding identifying nucleic acid fragments bound to the solid support, and the hybrids formed on the solid support are labeled with the known labeled nucleic acid fragments. A set of reagent combinations for use in the method according to claim 1, characterized in that the set comprises at least two nucleic acid reagents for each microbe and / or microbial group to be identified, for which the two different nucleic acid fragments derived from the microbe and / or microbial group are 63596 27 prepared from different parts of the microbial genome but preferably from closely spaced fragments, either directly from the microbial genome or by recombinant DNA techniques known per se, and these microbial-specific, either group- or species-specific nucleic acid fragments are single-stranded and one is attached to a solid support and the other labeled. A series of reagent combinations according to claim 2, characterized in that it comprises, as a nucleic acid reagent bound to a solid support, the adenovirus recombinant plasmid Ad2DpOR322 as a nucleic acid reagent and the labeled adenovirus AdI-BamHI C fragment as a labeled nucleic acid reagent. 15 A series of reagent combinations according to claim 2, characterized in that it comprises, as a nucleic acid reagent bound to a solid support, the plasmid pKTH312 for the identification of Semliki Forest virus. EcoRI-XhoI fragment A and EcoRI-XhoI fragment B of plasmid pKTH312 as labeled nucleic acid reagent. A series of reagent combinations according to claim 2, characterized in that the PstI B fragment of SV40 virus is used as a nucleic acid reagent bound to a solid support and the PstI A fragment of SV40 virus is labeled as a labeled nucleic acid reagent. A set of reagent combinations according to claim 2, characterized in that the identification of Bacillus amyloliquefaciens comprises, as a nucleic acid reagent bound to a solid support, an EcoRI-BamHIac The ClaI-EcoRI fragment of the CX amylase gene in plasmid pKTHIO 26 63596. A set of reagent combinations according to claim 2, characterized in that it comprises the plasmid pKTH45 as a nucleic acid reagent bound to a solid support for the identification of Escherichia coli and the recombinant phage mKTH1207 as a labeled nucleic acid reagent. B. A set of reagent combinations according to claims 2, 3, 5 and 6, characterized in that the microbes mentioned therein are capable of identifying and separating both viruses and bacteria such as adenovirus, SV40 virus and Bacillus amyloliquefaci from the same sample containing nucleic acids from various microbes include adenovirus Ad2DpBR322 recombinant plasmid, SV40 virus PstI B fragment, and the CX-amylase-20 gene of the Bacillus amyloliquefaciens plasmid pKTHIO plasmid EcoX-Bami Eco; and, as labeled nucleic acid reagents, the Ad2-BamHI C fragment of adenovirus, the PstI A fragment of SV40 virus, and the ClaI-EcoRI fragment of the CX amylase gene in the pKTHIO plasmid of Bacillus amylolique-faciens a. 2 '} Patentkrav 63 5 9 6